ETI signaling nodes are involved in resistance of Hawaii 7996 to Ralstonia solanacearum-induced bacterial wilt disease in tomato

ABSTRACT Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is a destructive disease of tomato. Tomato cultivar Hawaii 7996 is well-known for its stable resistance against R. solanacearum. However, the resistance mechanism of Hawaii 7996 has not yet been revealed. Here, we showed that Hawaii 7996 activated root cell death response and exhibited stronger defense gene induction than the susceptible cultivar Moneymaker after R. solanacearum GMI1000 infection. By employing virus-induced gene silencing (VIGS) and CRISPR/Cas9 technologies, we found that SlNRG1-silenced and SlADR1-silenced/knockout mutant tomato partially or completely lost resistance to bacterial wilt, indicating that helper NLRs SlADR1 and SlNRG1, the key nodes of effector-triggered immunity (ETI) pathways, are required for Hawaii 7996 resistance. In addition, while SlNDR1 was dispensable for the resistance of Hawaii 7996 to R. solanacearum, SlEDS1, SlSAG101a/b, and SlPAD4 were essential for the immune signaling pathways in Hawaii 7996. Overall, our results suggested that robust resistance of Hawaii 7996 to R. solanacearum relied on the involvement of multiple conserved key nodes of the ETI signaling pathways. This study sheds light on the molecular mechanisms underlying tomato resistance to R. solanacearum and will accelerate the breeding of tomatoes resilient to diseases.


Introduction
Ralstonia solanacearum as a soil-borne pathogen, can cause bacterial wilt in tomato, leading to huge economic losses worldwide every year. 1 R. solanacearum first colonizes the root surface and enters roots through natural openings or wounds. A multitude of plant cell-wall-degrading enzymes, as well as exopolysaccharides (EPS) and dozens of type III effectors, are subsequently secreted by the pathogen, which promote its proliferation into stem xylem vessels. The aerial parts of tomato plants are rapidly occupied by R. solanacearum, resulting in wilting and death. 2 Despite the application of diverse chemical or biological management strategies to control bacterial wilt of tomato, they appear to be less effective. 3 Therefore, disease resistance breeding is widely regarded as the best approach for controlling this devastating disease. 4 Solanum lycopersicum cultivar Hawaii 7996 is a stable resistant resource against diverse R. solanacearum strains under different environmental conditions. 5 Previous studies have shown that restricted shoot colonization of R. solanacearum in resistant tomatoes is attributed to limited root invasion, 6 which prevents the migration of the bacteria from the root collar to the midstem. 7 Grafting with Hawaii 7996 as the rootstock is one of the most efficient methods to constrain R. solanacearum invasion and hinder its spread into the scion. 8 The comparison of the transcriptional responses has revealed that resistant Hawaii 7996 roots initiated a stronger and earlier activation of defense-related genes than susceptible West Virginia 700 tomato roots upon inoculation with R. solanacearum. 9 The major quantitative trait loci (QTLs) for Hawaii 7996 resistance have been detected on chromosomes 12 and 6 (Bwr-12 and Bwr-6) from the recombinant inbred lines of Hawaii 7996 and West Virginia 700. 10,11 The analysis of genome-wide single-nucleotide polymorphisms (SNPs) revealed that 4 genes encoding putative leucine-rich repeat (LRR) receptor-like proteins were tightly linked to the chromosome 12. 12 However, the responsible genes contributing to Hawaii 7996 resistance have not been identified successfully yet.
Plants have evolved a layered innate immune system to fend off potential pathogens. On the one hand, cell-surface localized pattern recognition receptors (PRRs) can recognize pathogenrelated molecular patterns (PAMPs) or damage-related molecular patterns (DAMPs) to mount pattern-triggered immunity (PTI). 13 Receptor-like kinases (RLKs) or receptor-like proteins (RLPs) are representative members of plant PRRs. 14,15 A typical plant RLK contains an extracellular ectodomain, a singlepass transmembrane domain, and a cytoplasmic kinase domain, whereas an RLP largely resembles an RLK but lacks a cytoplasmic kinase domain. 16 Leucine-rich repeat (LRR) domain-containing RLKs and RLPs consist of the largest subfamily of plant PRR receptors. To transduce immune signals perceived by the ectodomains, LRR-RLP constitutively interacts with the LRR-RLK suppressor BIR1 (SOBIR1) through their transmembrane domains. In some cases, BRI1-ASSOCIATED KINASE-1 (BAK1) has been identified as a co-receptor indispensable for LRR-type PRR-mediated immunity. 17 On the other hand, plants employ intracellular nucleotide-binding leucine-rich repeat (NLR) proteins to detect pathogen effectors and activate effector-triggered immunity (ETI). 18,19 The NLR superfamily mainly consists of three subgroups: toll/interleukin-1 receptor NLRs (TNLs), helical coiled-coil NLRs (CNLs), and RPW8-like coiled-coil domain NLRs (RNLs). 20,21 ETI usually results in strong plant defense responses, or even local programmed cell death, to restrict pathogen colonization, which is known as the hypersensitive response (HR).
In Arabidopsis, Non-race-specific Disease Resistance 1 (NDR1) has been identified as a critical component in CNL signaling pathway. 22 ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), which is a lipase-like protein, is required for TNL-mediated resistance and can activate transcriptional defense response and HR. 23 In addition, another two EDS1 family proteins PHYTOALEXIN DEFICIENT 4 (PAD4) and SENESCENCE-ASSOCIATED GENE101 (SAG101) participate in basal resistance and ETI. 24,25 Helper NLRs, such as ACTIVATED DISEASE RESISTANCE 1 (ADR1)-related and N REQUIREMENT GENE 1 (NRG1)-related RNLs, play a critical role in TNL signal network in plants. Recent studies have indicated that NRG1/EDS1/SAG101 and ADR1/ EDS1/PAD4 are two distinct modules which cooperate to trigger immune signaling pathways. 26 Moreover, the mutual potentiation between PTI and ETI has been well documented, and LRR-RLP can mediate PTI responses in a EDS1/PAD4/ADR1 moduledependent manner. 21 Nevertheless, less is known about the potential effects of different immune pathways on the resistance of Hawaii 7996 against R. solanacearum.
In this study, we investigated the resistance mechanism of Hawaii 7996 against R. solanacearum. Using VIGS technology, we demonstrated that the immune signaling pathways depended on EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 nodules in the resistant cultivar Hawaii 7996. This study advances our understanding of molecular mechanism underlying resistance of Hawaii 7996 to R. solanacearum and offers new strategy for breeding disease-resistant crops against this devastating pathogen.

Bacterial strains
Ralstonia solanacearum strain GMI1000 was grown on CPG solid medium (1 g casamino acid, 10 g peptone, and 5 g glucose, 20 g agar, and 1 L water) at 28°C for 2 days and cultured in CPG liquid medium for 12 hours. Agrobacterium tumefaciens strain GV3101 carrying different constructs was incubated in LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl, and 1 L water) with 25 μg mL −1 gentamicin and respective antibiotics at 28°C for 12 hours.

Plasmid construction
The cDNA of Hawaii 7996 was used as a template to amplify the coding sequences of SlADR1, SlNRG1, SlEDS1, SlNDR1, SlPAD4, SlSAG101a, SlSAG101b, SlBAK1a, SlBAK1b, SlSOBIR1a, and SlSOBIR1b with the primers listed in Table S1. The amplified fragments were cloned into the pYL156 vector.

CRIPSR/Cas9 gene editing
The CRISPR/Cas9 binary vectors (pTX) were derived from pBin19, and the target sequence and Cas9 of CRISPR/Cas9 were driven by the tomato U6 promoter and 2 × 35S promoter, respectively. The recombinant pTX vector was designed to produce the deletions of SlADR1 coding sequence using one sgRNA alongside Cas9 endonuclease gene. The sgRNAs used in this study were presented in Table S1). By pTXCRISPR/Cas9-SlADR1 plasmid was transformed into Hawaii 7996 using the Agrobacterium-mediated transformation method. The transgenic tomato lines were selected based on their kanamycin resistance. Positive detection of each plant was conducted through PCR for verifying indel mutations on the left of sgRNA (Table S1).

RNA isolation, reverse transcription (RT), and quantitative PCR (qPCR) analysis
Using Trizol reagent from TIANGEN (Cat. DP424), total RNA was extracted from root tissues obtained from 2week-old Moneymaker and Hawaii 7996 plants grown on 1/2 MS medium or obtained from 4-week-old Hawaii 7996 plants grown in soil. The cDNA was synthesized using reverse transcript kit. Total RNA was treated with MonScript TM dsDNase for 2 min at 37°C, and then the first-strand cDNA was synthesized at 50°C for 30 min (Monad, Cat. MR05201). qPCR analysis was conducted using SYBR green Supermix (Monad, Cat. MQ00401S) with the gene-specific primers (Table S1) on the Analytikjena qTOWER 3 System following standard protocols. The expression of each gene was normalized to the expression of SlACTIN (Solyc11g005330.1). qPCR was performed with 1 μL cDNA reaction system with 32 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 50 s (Table S1). The melting curve was plotted from 65°C to 95°C ( Figure S1, S2, S4 and S5).

Virus-induced gene silencing in Hawaii 7996
Agrobacterium tumefaciens strain GV3101 carrying the tobacco rattle virus (TRV) constructs was cultured for the infiltration of Hawaii 7996. TRV-RNA1 cultures were mixed with equal volume of TRV-RNA2 cultures for silencing target genes. TRV-RNA2 vectors included TRV-green fluorescent protein (GFP), TRV-phytoene desaturase (PDS), SlBAK1b, TRV-SlBAK1a/b, TRV-SlSOBIR1a, TRV-SlSOBIR1b, and TRV-SlSOBIR1a/b. Twoweek-old Hawaii 7996 plants were infiltrated with A. tumefaciens cultures carrying the TRV constructs, and the silencing efficiency was assessed through the leaf chlorosis phenotype of TRV-SlPDS silencing. The infiltrated plants were grown indoors at 22°C with a 12 h light and 12 h dark photoperiod for 4 weeks, followed by inoculation assays.

ETI-related cell death is involved in Hawaii 7996-mediated resistance to R. solanacearum
Since Hawaii 7996-mediated resistance to bacterial wilt displayed pathogen strain specificity, we first examined whether HR-like cell death could occur during R. solanacearum GMI1000 infection. R. solanacearum bacterial infection assay was performed on two-week-old Hawaii 7996 seedlings grown in 1/2 MS plates with the susceptible cultivar Moneymaker as a control. After 48 hours post inoculation (hpi) Evans blue staining, the roots of Hawaii 7996 plants infected with R. solanacearum GMI1000 showed obvious cell death, but Moneymaker plant roots did not (Figure 1a). We further investigated the expression of ETI-related maker gene SlPR1b (Pathogenesis-related protein 1b) in tomato plant roots infected with GMI1000 and detected higher expression level of SlPR1b in Hawaii 7996 than in Moneymaker at 24 hpi (Figure 1b, Figure S1). Taken together, the above data indicated that Hawaii 7996 was resistant to R. solanacearum GMI1000 and elicited stronger ETI responses such as HRlike cell death than the susceptible cultivar upon R. solanacearum infection.   Figure S3). Next, we examined whether SlADR1 was required for the resistance of Hawaii 7996 against Bacterial growth in the inoculated tomato stems was quantified at 3 dpi from 6 technical replicates. Values represents means ± SD and asterisk indicates a significant difference with control difference (Student's one-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001). Values represents means ± SD and asterisk indicates a significant difference with control difference (Student's one-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001). Figure 3b: The box of the PAM sequence has shifted e2194747-4 GMI1000 through bacterial inoculation assay. The results showed that, in contrast to the WT plants, sladr1-1 mutant plants were more susceptible and developed disease symptoms much faster (Figure 3c-d). The mutant plants completely wilted at 7 dpi, whereas most of WT plants were healthy and showed resistance to GMI1000 infection (Figure 3e). Bacterial titers in sladr1-1 mutant plants were extraordinarily elevated in comparison to WT plants (Figure 3f). The above results indicated that sladr1-1 mutant plants completely lost the resistance to R. solanacearum GMI1000 infection.

Resistance of Hawaii 7996 is dependent on EDS1-related complex
In Arabidopsis, EDS1-PAD4-ADR1 complex is essential for TNLinduced basal resistance, whereas EDS1-SAG101-NRG1 complex is a critical component for TNL-mediated cell death. 27,29 Meanwhile, EDS1 and NDR1 are the conserved signaling nodes in TNL and CNL pathways, respectively. 23,30 To elucidate the roles of EDS1 and NDR1 in resistance of Hawaii 7996 to R. solanacearum, leaves of Hawaii 7996 plants were infiltrated with Agrobacterium carrying TRV2-SlEDS1 or TRV2-SlNDR1. The results showed that the expression of SlEDS1 and SlNDR1 was significantly reduced in corresponding gene-silenced plants (Figure 4a, Figure S4a-S4b). Three weeks after VIGS, the resistance of the gene-silenced plants to R. solanacearum GMI1000 was determined. The data showed that SlEDS1-silenced plants, but not SlNDR1-silenced plants, were more severe in disease symptoms ( Figure 4b) and disease index (Figure 4c) when compared to the TRV-GFP control plants. Additionally, a relatively lower survival rate was observed in SlEDS1-silenced plants than control plants at 11 dpi (Figure 4d). Bacterial titers in stems of tomato plants with TRV-SlEDS1 were 10 times higher than those of the control plants TRV-GFP (Figure 4e). Collectively, these results suggested that SlEDS1 is required for the resistance of Hawaii 7996 against R. solanacearum.
To investigate whether the other components in these signaling modules also participate in the resistance of Hawaii 7996, we performed VIGS to silence SlPAD4 or SlSAG101a/b in tomato plants, then challenged the plants with GMI1000. The qPCR results showed that the target genes were specifically silenced without affecting the expression of the close homologues in the EDS1 family (Figure 5a, Figure S4c 12). (e) Bacterial growth in the inoculated tomato stems was quantified at 3 dpi. Values represents means ± SD (n = 6 individual plants) and asterisk indicates a significant difference with control difference (Student's one-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).

PRR coreceptors contribute to Hawaii 7996 resistance
Considering that EDS1-PAD4-ADR1 is linked to RLP-SOBIR1 receptor complex, 21 we sought to figure out whether tomato PRR coreceptors BAK1 and SOBIR1 are involved in the resistance of tomato Hawaii 7996. Transient silencing of SlBAK1a and SlBAK1b encoding Arabidopsis BAK1 orthologues, or SlSOBIR1a and SlSOBIR1b encoding SOBIR1 orthologues were carried out by VIGS, in which these genes were specifically silenced in the corresponding plants (Figure 6a, Figure  S5). Then, we investigated the plant resistance against GMI1000 infection. Plants silencing SlBAK1a, SlBAK1b, SlSOBIR1a, or SlSOBIR1b individually did not show clear bacterial wilt symptom, compared with the control TRV-GFP plants ( Figure S6). However, the 25% of SlBAK1a/b-silenced plants and 37.5% of SlSOBIR1a/b-silenced plants displayed the enhanced susceptibility and faster disease symptoms than the control plants (Figure 6b-c). Meanwhile, we observed a reduced survival rate in TRV-SlBAK1a/b-or TRV-SlSOBIR1a/b-silenced plants infected with GMI1000 in contrast to TRV-GFP plants (Figure 6d). Bacterial titers in the TRV-SlBAK1a/b-or TRV-SlSOBIR1a/b-silenced plants at 3 dpi were significantly higher than those in the TRV-GFP plants (Figure 6e). Our data suggested that tomato BAK1 and SOBIR1 are partially required for the resistance of Hawaii 7996 to R. solanacearum.

Discussion
In this study, we compared the immune responses in resistant and susceptible tomato cultivars upon R. solanacearum infection, including defense-related gene expression and HR response. We found that induction of SlPR1b gene and HR response of the resistant cultivar Hawaii 7996 was faster and stronger than that of susceptible cultivar Moneymaker upon infection with R. solanacearum. By deploying VIGS and CRISPR-Cas9 technologies, we found that the resistance of Hawaii 7996 to R. solanacearum requires the involvement of multiple key components in the ETI signaling pathway ( Figure  S7). Our results offer valuable insights in understanding the disease resistance of tomato to bacterial wilt.
Hawaii 7996 has stable and robust resistance against various R. solanacearum in tomato and shows a high survival rate, but the corresponding molecular mechanism remains largely elusive. The explorations of the resistance phenotypes at the physiological, metabolism, and transcriptional levels have indicated that Hawaii 7996 can effectively limit the growth, colonization, and EPS production of R. solanacearum in vascular tissues. 6 Hawaii 7996 restricts the R. solanacearum infection by limiting bacterial root colonization, vertical movement upwards from root to the stem, the invasion into circular vascular bundles, and radial apoplastic spread in the cortex 8 . Immune gene activation is a rapid and effective defense Bacterial growth in the inoculated tomato stems was quantified at 3 dpi from 6 technical replicates. Values represents means ± SD and asterisk indicates a significant difference with control difference (Student's one-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).
response for plants against pathogens. 9,31,32 Cell death or HR occurs in resistance cultivar LS-89 after R. solanacearum infection. 33 Similarly, this study discovered that R. solanacearum GMI1000 induced cell death in the roots of Hawaii 7996, which was not observed in Moneymaker. In addition, the induction of immune-related gene SlPR1b in Hawaii 7996 was much stronger after GMI1000 inoculation, compared with that in Moneymaker, implying that the strength of ETI was associated with tomato resistance to bacterial wilt.
Further, we revealed that the key node components in the ETI signaling pathway were required for Hawaii 7996 resistance, thereby expanding our knowledge of tomato resistance to R. solanacearum. In Arabidopsis, TNL pair RRS1/RPS4 can recognize AvrRps4 from Pseudomonas syringae and PopP2 from R. solanacearum. Helper NLRs was required for multiple sensor NLRs to mediate downstream signaling. For instance, RRS1/ RPS4-mediated bacterial resistance requires the participation of RNLs including NRG1A, NRG1B, and ADR1s. [34][35][36] However, RRS1/RPS4-mediated HR is dependent on NRG1s but not ADR1s. 27 In this study, SlADR1-and/or SlNRG1-silenced Hawaii 7996 plants exhibited impaired resistance to R. solanacearum. In addition, our results showed that sladr1-1 mutant plants completely lost the resistance to GMI1000. These data jointly indicated that helper NLRs ADR1 and NRG1 are involved in Hawaii 7996-mediated resistance to R. solanacearum. NDR1 and EDS1 are conserved critical nodes required for ETI activation. Our data showed that the resistance of Hawaii 7996 was independent of SlNDR1, but dependent on SlEDS1, which suggested TNLs likely play critical roles in the resistance of Hawaii 7996. Meanwhile, we found that SlPAD4 and SlSAG101a/b are both required to mount effective resistance in Hawaii 7996. In Arabidopsis, EDS1-PAD4-ADR1 complex is required for TNL-induced basal resistance, whereas EDS1-SAG101-NRG1 complex is a critical component for TNLmediated cell death. 27,29 ADR1 and NRG1 participate in plant immunity as helper NLRs. 34 Our data indicated that both EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 modules are strictly required for the resistance of Hawaii 7996 toward R. solanacearum. It is noteworthy that PTI and ETI do not operate independently but rather exhibit mutual potentiation, and EDS1-PAD4-ADR1 signaling module is required for RLPmediated resistance. 37,38 In this study, we discovered that silencing SlBAK1a/b or SlSOBIR1a/b resulted in partial loss of resistance of Hawaii 7996 plants to wilt disease, indicating that certain PRR(s) might participate in this resistance. Further, QTLs related to R. solanacearum resistance were mapped onto chromosome 12, which contains multiple RLP genes. 12 The exact roles of those candidate RLPs in the resistance against R. solanacearum awaits future investigation. The key receptors recognizing R. solanacearum in both PTI and ETI (e) Bacterial growth in the inoculated tomato stems was quantified at 3 dpi. Values represents means ±SD (n = 6 individual plants) and asterisk indicates a significant difference with control difference (Student's one-tailed ttest, * p < 0.05, ** p < 0.01, *** p < 0.001). The above experiments were repeated at least three times with similar results. pathways in Hawaii 7996 require further exploration. The related findings will aid in understanding the resistance mechanisms of Hawaii 7996 to R. solanacearum and provide new insights and solutions to disease resistance breeding of tomato.

Disclosure statement
No potential conflict of interest was reported by the authors.

Author contributions
B.L. designed and coordinated the entire project. A.X. performed experiments, data analysis and wrote the manuscript. L.W. helped with the VIGS assay. J.K. and C.F. generated the tomato mutant. P.L., C.F., and X.Y. analyzed data and provided critical feedback. B.L., A.X., and X.Y. participated in manuscript revision. All authors read and approved the manuscript.